Formation of bone tissue requires transcriptional mechanisms to induce a program of gene expression for progressive development of the osteoblast phenotype. During osteoblast differentiation temporally expressed genes are induced and repressed at specific stages. Transcription factors will interact with co-regulatory proteins to provide multiple options for positive and negative gene specific transcriptional control. We have shown that three principal classes of transcription factors mediate activation of the bone specific osteocalcin (OC) gene in vitro, however, there is no understanding of their regulatory interplay in control of bone specific gene expression in vivo and osteoblast differentiation. Based on our discoveries, we hypothesize that in addition to the Runx2/Cbfal transcription factor, homeodomain and C/EBP proteins together with their co-regulatory factors, support osteoblast differentiation by regulating the induction/repression of bone specific genes during stages of osteoblast differentiation in vivo through formation of temporally and functional distinct protein-DNA and protein-protein interactions to control transcription. We are addressing how transcription brings about expression of the osteoblast phenotype through analysis of the OC gene promoter. Using mouse models, functions of the homeodomain (Aim 1) and C/EBP (Aim 2) transcription factors in regulating developmental and tissue restricted OC expression and in supporting osteoblast differentiation will be investigated. We will address when in vivo, specific members of the homeodomain and C/EBP families occupy their regulatory elements during osteoblast differentiation (Aim 3). The functional activity of chromatin modifying co-regulatory proteins in supporting osteoblast specific gene expression will be determined (Aim 4). These studies will establish the importance of bone selective transcription factor complexes for formation, maintenance and remodeling of bone tissue.